Known single molecule sensors typically require a fluorescent or metallic label that is attached to the target molecule so that the target molecule can be identified. Such labels, however, require prior knowledge of the presence of the target molecule. Thus, known sensor systems that require labels are not suitable for blind detection of target molecules, which do not have labels. Further, such labels may require additional data processing. For example, sensors using labels may require ensemble averaging of large numbers of cells, thereby resulting in confusion or dulling of recorded responses in those cases in which there is any heterogeneity in the cells or their responses. As a result, detection using labels cannot be performed in real-time.
Several devices have been used for label-free detection including fiber optic waveguides, nanowires, nanoparticle probes, biochips, mechanical cantilevers and micro-sphere resonators. For example, U.S. Pat. No. 4,071,753 to Fulenwider et al. and U.S. Pat. No. 4,419,895 to Fuller describe optical fiber sensors. In such systems, the optical coupling coefficient between two fibers varies with the physical parameter to be measured, so that by measuring such coefficient, the parameter can be detected and measured. In another type of optical sensor, the physical parameter to be measured modulates the vibrational motion of a transducer. Such modulation changes the intensity of light coupled between the ends of two optical fibers so that by measuring such changes the physical parameter can be detected and measured. U.S. Pat. No. 6,583,399 to Painter et al. describes a micro-sphere resonant sensor that includes a modifier that is bound to an outer surface of the spherical resonator. The modifier provides a binding site such that a binding event occurs at the outer surface of the micro-sphere in the presence of a target molecule. While certain known devices may provide label-free detection, they have a number of limitations and can be improved.
Initially, various known sensors lack the sensitivity to allow detection of a very small number of molecules or a single molecule and, therefore, may not be suitable for biological and chemical analyses requiring more specific detection such as cell signaling and cellular dynamics. For example, single molecule detection capabilities are increasingly important for biologists and chemists who are working to unravel the complex nature of cell signaling and cellular dynamics, e.g., monitoring biochemical pathways at the single cell level. Single molecule detection can be used in various environmental applications. In contrast, previous experiments with silica micro-spheres, for example, demonstrated gross detection of approximately 1 billion molecules. Accordingly, such devices are not suitable for detection of a very small number of molecules or a single molecule.
The reasons for inadequate sensitivities are specific to each type of sensor. For example, sensitivities of sensors having mechanical components may be limited by the sensitivities that can be achieved given the particular mechanical construct. Mechanical resonators have demonstrated single molecule sensitivity at liquid nitrogen/liquid helium temperatures. However, such capabilities are not suitable for biological detection because molecules of interest do not exist in their nature conformations at these temperatures. Further, such devices are often subject to electromagnetic interference. In the case of certain optical sensors and traps, sensitivity limitations are due, in part, to the limited interaction of light with the target molecule. For example, in a simple optical waveguide sensor, the input light has only one opportunity to interact with the target molecule.
Various sensors also present manufacturing and integration challenges, which limit the extent to which such devices can be used on a large-scale basis. For example, known micro-sphere resonators are typically limited to laboratory applications and experiments as a result of their spherical shape and the fabrication controls that are needed to produce such shapes. Additionally, certain devices have been characterized in an air environment, but nearly all molecular detections are performed in a solution or liquid.
Further, in the case of optical sensors, it is necessary to increase the evanescent field intensity to increase the detection limit into the single molecule regime, which many optical sensors cannot do. Increases in evanescent field and detection sensitivity were demonstrated previously using a levitated micro-droplet resonator formed from various liquids. However, such micro-droplet resonators are not practical since they cannot be immersed in liquid and require fluorescent labels and magneto-optical traps to maintain their spherical shape.
In addition to challenges of detection a small number of molecules or single molecule, known devices also have limitations when detecting a single species in a mixture of chemically similar molecules. Several different systems and techniques have been used for this purpose including spectroscopic (emission and absorption) techniques proton conductors and nuclear magnetic resonance, but known systems and techniques also have limited sensitivities. For example, known sensors are only capable of detecting 30 parts per million per volume (ppmv) of heavy water (D2O) in water (H2O). Such capabilities may not be sufficient in applications requiring more sensitive heavy water detection capabilities in order to detect removal of naturally occurring heavy water from public water sources or to identify potential nuclear activities.
Accordingly, blind, label-free molecule detection methods and sensor devices having enhanced specificity and sensitivity to detect a very small number of molecules or a single molecule would be desirable. It would also be desirable to detect a single species in a mixture of chemically similar molecules. It would also be desirable to have sensors that interact with molecules more than only one time as in known various known optical waveguides. Further, it would be desirable to have label-free sensor devices that can be manufactured and implemented more easily than other devices that present manufacturing and integration challenges. It would also be desirable to have label-free detection methods and sensors in order to enable new biological, chemical, biochemical and environmental research and applications. It would also be desirable to have detection methods and sensor devices that can operate in different environments. Moreover, it would be desirable to have label-free sensors that can be functionalized to detect a small number of molecules or a single molecule of various types for use in different applications.